[0001] The present invention relates to a novel process for preparing certain griseolic
acid derivatives, which process enables the derivatives to be prepared in a high yield
and by means of a simple process.
[0002] Griseolic acid is a nucleoside-type compound having an adenine base and two carboxylic
acid groups. It was first disclosed in, inter alia, European Patent Specification
No. 29,329A, and subsequently its structure was disclosed in US Patent Specification
No. 4,460,765.
[0003] Subsequently, copending European Patent Publication No. 0143557, disclosed a class
of griseolic acid derivatives having activities at least as good as the natural product,
griseolic acid, but having significantly lower toxicities. Certain of the compounds
prepared by the process of the present invention are embraced by the general disclosure
of said European Patent Publication, although only one such compound, N
6-methylgriseolic acid, is specifically disclosed. Other compounds prepared by the present
invention are disclosed in European Patent Application No. 86303000.3.
[0004] The general class of griseolic acid derivatives, and especially the N
6-alkyl and N
6-aralkyt griseolic acid derivatives, are non-toxic and have a variety of valuable therapeutic
activities arising primarily from their abilities to inhibit the activity of phosphodiesterases
(PDE) of, for example, cyclic adenosine monophosphate (cAMP). For example, they have
shown the potential to be used as ameliorators of cerebral function, as angiocar-
diokinetic agents, as antithrombotic agents, as diuretics, as psychotropic and neurotropic
agents, as smooth muscle relaxants and as anticancer agents.
[0005] However, it is always necessary that drugs should be capable of preparation at a
cost at most commensurate with their therapeutic value and preferably as cheaply as
possible. In any case, regardless of cost, where a drug is prepared by a multi-stage
sequence of reactions or in low yield, there is always an increased danger that the
drug may be contaminated by by-products which may be difficult or impossible to remove,
and its value may thereby be degraded.
[0006] We have now discovered a process for preparing certain griseolic acid derivatives,
which process allows the derivatives to be obtained in a high yield and in very few
reaction steps.
[0007] The compounds prepared by the process of the present invention are those compounds
of formula (I):

wherein:
R' represents a hydrogen atom or a hydroxy-protecting group;
R2 represents a hydrogen atom, a hydroxy group or a protected hydroxy group;
R3 and Fr are independently selected from the group consisting of hydrogen atoms and carboxy-protecting
groups;
R5 and R6 each represent hydrogen atoms or together represent an extra carbon-carbon bond between
the carbon atoms to which they are attached; and
R' represents an alkyl group or an aralkyl group;
and salts thereof.
[0008] The process of the present invention comprises reacting a compound of formula (II):

(in which R', R
2, R', R
4, R
5 and R
6 are as defined above) with a compound of formula (III):

[0009] (in which: R
7 is as defined above; and X represents a halogen atom, a C
1-C
6 alkylsulfonyloxy group, a fluorinated C
1-C
6 alkylsulfonyloxy group, an arylsulfonyloxy group or a C
1-C
6 alkoxysulfonyloxy group), subjecting the aminopyrimidine ring of the product to ring
cleavage, rearrangement and ring formation and, if desired, removing any protecting
group and, if desired, salifying the product.
[0010] The process of the present invention may be represented by the reaction shown in
the following scheme:

(in which R
1-R
7 and X are as defined above) followed, if necessary, by removal of protecting groups
and/or salification.
[0011] Where R', R
2, R' or R
4 represents a protecting or protected group, the nature of such a group is not critical
to the present invention. Where the final products of formula (I) are themselves to
be used directly for therapeutic purposes, and where such a protecting group remains
in the product of formula (I), it is, of course, necessary that the protecting group
should not adversely affect the therapeutic value of the compounds (for example by
either reducing their activity or increasing their toxicity to an unacceptable extent).
However, where protecting groups are employed which do have such an adverse effect,
they may easily be removed in the final step of the reaction sequence (as described
in more detail hereafter). Alternatively, the protecting groups employed may be pharmaceutically
acceptable and, in that case, the resulting compounds of formula (I) may be used directly
for therapeutic purposes. Also, of course, if the compounds of formula (I) are themselves
to be employed as intermediates in the preparation of yet other griseolic acid derivatives,
it may be unnecessary to remove protecting groups and the nature of such protecting
groups will not be critical.
[0012] Where R' or R
2 represents, respectively, a hydroxy-protecting group or a protected hydroxy group,
examples of suitable hydroxy-protecting groups include aliphatic acyl groups, aromatic
acyl groups, heterocyclic groups, tri-substituted silyl groups, lower alkyl groups,
lower alkoxymethyl groups, substituted ethyl groups, aralkyl groups, lower alkoxycarbonyl
groups, lower alkenyloxycarbonyl groups and protecting groups which are easily hydrolized
in vivo.
[0013] Where the hydroxy-protecting group is an aliphatic acyl group, this is a carboxylic
acyl group preferably having up to 6 carbon atoms and is more preferably an alkanoyl
or alkenoyl group having up to 6, preferably up to 5, carbon atoms, which group may
be unsubstituted or may have at least one substituent selected from the group consisting
of halogen atoms, C,-C4 alkoxy groups and aryloxy groups. Examples include the acetyl,
chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, methoxyacetyl, propionyl,
butyryl, - (E)-2-methyl-2-butenoyl, isobutyryl, valeryl, isovaleryl and pivaloyl groups.
[0014] Where the hydroxy-protecting group is an aromatic acyl group, this is preferably
an arylcarbonyl group, where the aryl part is preferably a C6-C,,, more preferably
C
6-C
10, carbocyclic aryl group, which may be unsubstituted or may have at least one substituent
selected from the group consisting of halogen atoms, C
1-C
4 alkyl groups, C
1-C
4 alkoxy groups, aryl groups (which themselves are as defined in relation to the aryl
groups of these arylcarbonyl groups), C,-C4 haloalkyl groups, C
2-C
5 alkoxycarbonyl groups, cyano groups and nitro groups. Examples of such aromatic acyl
groups include the benzoyl, o-(dibromomethyl)benzoyl, o-(methoxycarbonyl)benzoyl,
p-phenylbenzoyl, 2,4,6-trimethylbenzoyl, p-toluoyl, p-anisoyl,
Q-chlorobenzoyl, p-nitrobenzoyl, Q-nitrobenzoyl and a-naphthoyl groups.
[0015] Where the hydroxy-protecting group is a heterocyclic group, this is preferably such
a group containing a single oxygen or sulfur hetero-atom and having 5 or 6 ring atoms
(including the hetero-atom), for example a tetrahydrofuryl, tetrahydrothienyl, tetrahydropyranyl
or tetrahydrothiopyranyl group. This may be unsubstituted or may have at least one
substituent selected from the group consisting of the substituents defined above in
relation to substituents on aryl groups, preferably halogen atoms or C
1-C
4 alkoxy groups. The tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuryl and
tetrahydrothienyl groups are preferred. Examples of such groups include the tetrahydropyran-2-yl,
3-bromotetrahydropyran-2-yl, 4-methoxytetrahydropyran-4-yl, tetrahydrothiopyran-2-yl
and 4-methoxytetrahydrothiopyran-4-yl groups.
[0016] Where the hydroxy-protecting group is a tri-substituted silyl group, the substituents,
which may be the same or different, are preferably C,-C4 alkyl groups and examples
of such substituted silyl groups include the trimethylsilyl, triethylsilyl, isopropyldimethylsilyl,
t-butyldimethylsilyl, diisopropylmethylsilyl, methyldi-t-butylsilyl and triisopropylsilyl
groups.
[0017] Where the hydroxy-protecting groups are lower alkyl groups, these preferably have
from 1 to 6 carbon atoms and may be straight or branched chain groups. Examples include
the methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl
and hexyl groups.
[0018] Where the hydroxy-protecting group is a lower alkoxymethyl group, this may be a mono-alkoxymethyl
or di-alkoxymethyl group and the alkoxy part is preferably a C
1-C
6 more preferably C
1-C
4, alkoxy group which may be unsubstituted or have at least one substituent selected
from the group consisting of C,-C4 alkoxy groups and halogen atoms. Examples of such
alkoxymethyl groups include the methoxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl,
2,2,2-trichloroethoxymethyl and bis(2-chloroethoxy)methyl groups.
[0019] Where the hydroxy-protecting group is a substituted ethyl group, the ethyl group
may have one or more, preferably from 1 to 3, substituents selected from the group
consisting of C,-C4 alkoxy groups, C
1-C
4 alkyl groups, halogen atoms, C,-C4 alkylselenyl and arylselenyl groups (in which
the aryl part is as defined in relation to the aryl parts of arylcarbonyl groups above).
Examples include the , 1-ethoxyethyl, 1-methyl-1-methoxyethyl, 1-isopropoxyethyl,
22,2-trichloroethyl and 2-phenyl- selenylethyl groups.
[0020] Where the hydroxy-protecting group is an aralkyl group, this may be a monoaryl-alkyl
group, a diaryl-alkyl group or a triaryl-alkyl group. Each aryl part is preferably
a C
6-C
14, more preferably C
6-C
10, carbocylic aryl group and is as defined above in relation to the aryl groups of
arylcarbonyl groups. It is preferably unsubstituted or has at least one substituent
selected from the group consisting of C
1-C
4 alkyl groups, C
1C
4 alkoxy groups, halogen atoms, nitro groups and cyano groups. The alkyl part is a
straight or branched chain group preferably having from 1 to 4, more preferably 1
to 3 and most preferably 1 or 2, carbon atoms. Examples of such aralkyl groups include
the benzyl, p-methoxybenzyl, o-nitrobenzyl, P-nitrobenzyl, P-halobenryl (where the
halo is preferably chloro, iodo, bromo or fluoro), P-cyanobenzyt, diphenylmethyl,
triphenylmethyl, α-naphthyldiphenylmethyl and p-methoxyphenyldiphenylmethyl groups.
[0021] Where the hydroxy-protecting group is a lower alkoxycarbonyl group, this is preferably
a C
2-C
7, more preferably C
2-C
5, alkoxycarbonyl group (i.e. the alkoxy part is C
1-C
6, more preferably C
1-C
4) and the alkoxy part may be unsubstituted or may have at least one substituent selected
from the group consisting of halogen atoms and tri-substituted silyl groups (e.g.
as defined above in relation to the substituted silyl groups which may act as hydroxy-protecting
groups). Examples of such alkoxycarbonyl groups include the methoxycarbonyl, ethoxycarbonyl,
t-butoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, isobutoxycarbonyl and 2-trimethylsilylethoxycarbonyl
groups.
[0022] Where the hydroxy-protecting group is a lower alkenyloxycarbonyl group, the alkenyl
part is preferably a C
2-C
4 alkenyl group and examples include the vinyloxycarbonyl and allyloxycarbonyl groups.
[0023] Where the hydroxy-protecting group is a group which is easily hydrolized in vivo,
the group may fall within a number of different classes, including some classes which
overlap with those hydroxy-protecting groups described above. In general, preferred
such hydroxy-protecting groups include: the aralkyloxycarbonyl groups (in which the
aralkyl part may be as defined above in relation to aralkyl groups which themselves
serve as hydroxy-protecting groups), for example the benzyloxycarbonyl, p-methoxybenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, o-niirobenzyloxycarbonyl and p-nitrobenzyloxycarbonyl
groups; and the acyloxy- substituted alkoxycarbonyl, preferably methoxycarbonyl, groups,
such as the pivaloyloxymethoxycarbonyl group.
[0024] Of these, we prefer the aliphatic acyl groups, the aromatic acyl groups and those
protecting groups which are easily hydrolized in vivo.
[0025] Where R
3 or R
4 represents a carboxy-protecting group, examples include lower alkyl groups, lower
haloalkyl groups, aralkyl groups and carboxy-protecting groups which are easily hydrolized
in vivo.
[0026] Where R
3 or R
4 represents a lower alkyl group, this has from 1 to 6, preferably from 1 to 4, carbon
atoms and may be a straight or branched chain group. Examples include the methyl,
ethyl, propyl, isopropyl, butyl, isobutyl and t-butyl groups.
[0027] Where R
3 or R
4 represents a lower haloalkyl group, this likewise has from 1 to 6, preferably from
1 to 4, carbon atoms and at least one halogen substituent, although more halogen substituents
may be present, up to complete perhalogenation. Examples include the 2,2,2-trichloroethyl,
2-chloroethyl, 2-bromoethyl, 2-iodoethyl, 2-fluoroethyl and 2,2-dibromoethyl groups.
[0028] Where R
3 or R
4 represents an aralkyl group, this may be a monoaryl-alkyl, diaryl-alkyl or triaryl-alkyl
group, preferably a monoaryl-alkyl or diaryl-alkyl group. The aryl part is preferably
a C
6-C
14 carbocyclic aryl group which may be unsubstituted or may have at least one substituent
selected from the group consisting of nitro groups, C
1-C
4 alkyl groups, C,-C4 alkoxy groups, halogen atoms and C, or C
2 alkylenedioxy groups. Examples of such aralkyl groups include the benzyl, p-rtitrobenzyt,
o-nitrobenzyl, triphenylmethyl, diphenylmethyl, bis(o-nitrophenyl)methyl, 9-anthrylmethyl,
2,4,6-trimethylbenzyl, p-bromobenzyl, g -methoxybenzyl, 3,4,5-trimethoxybenzyl and
piperonyl groups.
[0029] Where R
3 or R
4 represents a carboxy-protecting group which can easily be hydrolized in vivo, these
may be selected from a wide range of different classes of groups, which are well-known
to those skilled in the art. Examples include: aliphatic acyloxymethyl groups, in
which the aliphatic acyl part is preferably a C
2-C
6 alkanoyl group, for example the acetoxymethyl, propionyloxymethyl, butyryloxymethyl
or pivaloyloxymethyl groups; alkoxymethyl groups, where the alkoxy part is a C,-C
5, preferably C
1-C
4, alkoxy group, and may be unsubstituted or have at least one substituent (if substituted,
the substituent is preferably a single C
1-C
4 alkoxy substitutent) and examples of such alkoxymethyl groups include the methoxymethyl,
ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl and 2-methoxyethoxymethyl
groups; 1-alkoxycarbonyloxyethyl groups in which the alkoxy part is a C,-C
6, preferably C,-C,, alkoxy group, for example the 1-methoxycarbonyloxyethyl, 1- ethoxycarbonyloxyethyl,
1-propoxycarbonyloxyethyl, 1-isopropoxycarbonyloxyethyl, 1-butoxycarbonyloxyethyl
or 1-isobutoxycarbonyloxyethyl groups; the phthalidyl group; and the (2-oxo-5-methyl-1,3-dioxolen-4-yl)methyl
group.
[0030] Of these carboxy-protecting groups, the alkyl groups, aralkyl groups and groups which
are easily hydrolized in vivo are particularly preferred.
[0031] Where R' represents a lower alkyl group or an aralkyl group, these may be as defined
above in relation to the equivalent carboxy-protecting groups which may be represented
by R' or R4.
[0032] X represents a halogen atom, a lower alkylsulfonyloxy group, a fluorinated lower
alkylsulfonyloxy group, an arylsulfonyloxy group or a lower alkoxysulfonyloxy group.
[0033] Where X represents a halogen atom, this is preferably an iodine, chlorine or bromine
atom.
[0034] Where X represents a lower alkylsulfonyloxy group, this is a C
1-C
6, preferably C
1-C
4, alkanesul- fonyloxy group, for example a methanesulfonyloxy, ethanesulfonyloxy or
propanesulfonyloxy group.
[0035] Where X represents a fluorinated lower alkylsulfonyloxy group, this is an alkylsulfonyloxy
group as defined above which has at least one fluorine substituent, but which may
have more than one fluorine substituent and may be, and preferably is, completely
perfluorinated. Examples include the trifluoromethanesulfonyloxy and pen- tafluoroethanesulfonyloxy
groups.
[0036] Where X represents an arylsulfonyloxy group, the aryl part may be as defined above
in relation to the aryl part of the arylcarbonyl group which serves as a hydroxy-protecting
group, and examples include the benzenesulfonyloxy and p-toluenesul- fonyloxy groups.
[0037] Where X represents an alkoxysulfonyloxy group, the alkoxy part is a C
1-C
6, preferably C
1-C
4, alkoxy group and examples include the methox- ysulfonyloxy, ethoxysulfonyloxy and
propoxysul- fonyloxy groups.
[0038] We particularly prefer that X should represent a halogen atom.
[0039] In Step (a) of the process of the invention, the griseolic acid or derivative thereof
of formula (II) is subjected to alkylation or aralkylation with an alkylating or aralkylating
agent (III) in an inert solvent. The nature of the solvent is not particularly critical,
provided that it has no adverse effect upon the reaction. Examples of suitable solvents
include: alcohols, such as methanol, ethanol, isopropanol, butanol, and t-butanol;
ethers, such as diethyl ether, tetrahydrofuran, dioxane or ethylene glycol dimethyl
ether; nitriles, such as acetonitrile; amides, such as dimethylformamide, dimethylacetamide
or hexamethylphosphoric triamide; and sulfoxides, such as dimethyl sulfoxide. The
preferred solvents are amides or sulfoxides.
[0040] The reaction will take place over a wide range of temperatures, but we prefer to
carry out the reaction at a temperature of from 0°C to 100°C, more preferably from
room temperature to 70°C.
[0041] The time required for the reaction may vary widely, depending upon many factors,
notably the reaction temperature and the natures of the solvents and reagents employed.
In general, a period of from 30 minutes to 10 days will suffice. If, for example,
the reaction is carried out at room temperature, it is generally complete within from
1 to 7 days; on the other hand, at 70°C, it will normally be complete within from
1 to 20 hours.
[0042] The intermediate product of formula (II') may be obtained from the reaction mixture
by evaporating off the solvent under reduced pressure and then the product may be
subjected to Step (b) without further isolation, in the same reaction vessel. Alternatively,
if desired, the intermediate (11') may be isolated by conventional means before being
subjected to Step (b).
[0043] In Step (b), the compound is subjected to a ring-opening, rearrangement and ring-closure
reaction involving the pyrimidine ring and the free amino group.
[0044] In this step, the residue obtained from the alkylation or aralkylation reaction of
Step (a) is dissolved or suspended in a suitable solvent and the pH of the resulting
solution or suspension is adjusted or maintained at a value not less than 4, to effect
the aforesaid ring-opening, rearrangement and ring-closure reactions. The pH value
employed for these reactions is more preferably at least 5 and still more preferably
at least 7.
[0045] Maintenance of the chosen pH value may be achieved, for example, either (1) by conducting
the reactions in a buffer solution previously adjusted to an appropriate pH value
or (2) by standing or heating the residue in an excess of an aqueous solution of an
alkali metal or alkaline earth metal hydroxide or a solution containing an organic
base in water or in a suitable organic solvent.
[0046] There is no particular limitation upon the nature of the buffer solution to be employed,
provided that it is capable of maintaining an appropriate pH value throughout the
reaction of Step (b). Any conventional buffer solution, for example an acetate, phosphate,
borate, ammonium bicarbonate, phthalate or citrate buffer, may be used.
[0047] Examples of suitable alkali metal and alkaline earth metal hydroxides which may be
used in the aqueous solution include sodium hydroxide, potassium hydroxide, lithium
hydroxide and calcium hydroxide. Examples of suitable organic bases include, for example,
lower alkylamines, such as monomethylamine, dimethylamine or trimethylamine.
[0048] In geneal, the pH of the reaction solution is preferably maintained within the range
from 4 to 12, although higher pH values may also be employed.
[0049] There is no particular limitation on the nature of the solvent employed in this reaction,
provided that it does not interfere with the reactions. Suitable solvents include,
for example: water; alcohols, such as methanol, ethanol or propanol; and other water-
miscible solvents, such as acetone, tetrahydrofuran, dioxane, dimethylformamide or
dimethyl sulfoxide. A single such solvent or a mixture or any two or more thereof
may be employed. In some cases, the organic base may also act as the reaction solvent.
[0050] The reaction may take place over a wide range of temperatures, for example from 0°C
to 150°C, more preferably from 20°C to 100°C. The temperature chosen may depend upon
various factors. For example, heating may be preferable when the reaction is carried
out at a pH value within the range from 4 to 10; on the other hand, the reaction will
generally proceed satisfactorily at ambient temperature at a pH of 10 or above.
[0051] The time required for the reaction may vary widely, depending upon many factors,
notably the nature of the substrates, the reaction temperature and the pH and nature
of the buffer or other medium used, especially the temperature and pH; however, within
the preferred ranges indicated above, a period of from 5 minutes to 50 hours will
normally suffice.
[0052] After completion of the reaction, the resulting compound of formula (1) may be recovered
from the reaction mixture by conventional means, for example any one or any appropriate
combination of the following steps: adjustment of the pH of the reaction mixture;
concentration of the reaction mixture, e.g. by evaporating off the solvent under reduced
pressure; separating, e.g. by filtration, of the precipitate obtained from recrystallization
of the reaction residue;. or, if no crystalline precipitate is thereby produced, extracting
the mixture with a water-immiscible solvent and then evaporating the solvent from
the exiract. If desired, the resulting product may be further purified by conventional
techniques, for example recrystallization or the various chromatography techniques
such as column chromatography or preparative thin layer chromatography.
[0053] Where the resulting compound of formula (1) contains a hydroxy-protecting group and/or
a carboxy-protecting group, such protecting groups may, if desired, be removed in
a step following Step (b). The nature of the process employed to remove these protecting
groups will vary depending upon the nature of the protecting group, as is well-known
in the art.
[0054] For example, where a trialkylsilyl group is employed as a hydroxy-protecting group,
it may be removed by treating the compound of formula (I) with a compound which produces
fluoride anions, for example tetrabutylammonium fluoride. The reaction is preferably
effected in the presence of a solvent, the nature of which is not critical, provided
that it has no adverse effect upon the reaction; suitable solvents include ethers,
such as tetrahydrofuran or dioxane. The reaction temperature is not particularly critical,
and we generally find it convenient to carry out the reaction at about room temperature,
at which temperature a period of from 10 to 18 hours is normally required.
[0055] Where an aralkyloxycarbonyl group or aralkyl group is employed as a hydroxy-protecting
group, it may be removed by contacting the compound with a reducing agent. Suitable
reducing agents include, for example, hydrogen in the presence of a catalyst (e.g.
palladium-on-activated carbon or platinum) or an alkali metal sulfide (such as sodium
sulfide or potassium sulfide). These reactions are preferably carried out in the presence
of a solvent, the nature of which is not critical, provided that it has no adverse
effect upon the reaction. Examples of preferred solvents include: alcohols, such as
methanol or ethanol; ethers, such as tetrahydrofuran or dioxane; fatty acids, such
as acetic acid; and mixtures of one or more of these organic solvents with water.
The reaction will take place over a wide range of temperatures, the preferred temperature
depending upon the nature of the reducing agent. For example, in the case of catalytic
reduction, room temperature is preferred. In the case of reduction employing alkali
metal sulfides or similar reducing agents, a temperature of about room temperature
or below, e.g. down to 0°C, is normally preferred. The time required for the reaction
may vary widely, depending upon many factors, notably the natures of the starting
materials and reducing agents, and the reaction temperature; however, a period of
from 5 minutes to 12 hours will normally suffice.
[0056] Where a lower alkyl group, lower aliphatic acyl group, aromatic acyl group or alkoxycarbonyl
group is employed as the hydroxy-protecting group, it may be removed by treating the
compound of formula (I) with a base in the presence of an aqueous solvent. There is
no particular limitation on the nature of the solvent to be employed and any solvent
commonly used in hydrolysis reactions may equally be used in this reaction. Examples
of preferred solvents include water itself and mixtures of water with one or more
organic solvents, for example: alcohols, such as methanol, ethanol or propanol; and
ethers, such as tetrahydrofuran or dioxane. Equally, there is no particular limitation
on the nature of the base to be employed, and any base commonly used in hydrolysis
reactions may be employed, provided that it does not adversely affect other functional
groups of the compound. Examples of preferred bases include: alkali metal carbonates,
such as sodium carbonate or potassium carbonate; and ammonia in water or in a suitable
organic solvent. The reaction will take place over a wide range of temperatures, but
we generally prefer to employ a temperature of about room temperature or below, e.g.
down to 0°C. The time required for the reaction will vary, depending upon many factors,
notably the nature of the starting materials and the reaction temperature, but a period
of from 1 to 6 hours will normally suffice.
[0057] Where a heterocyclic group (e.g. a tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuryl
or tetrahydrothienyl group), an alkoxymethyl group or a substituted ethyl group is
employed as the hydroxy-protecting group, it may be removed by treating the compound
of formula (I) with an acid in a solvent. There is no particular limitation on the
nature of the acid and examples include hydrochloric acid, a mixture of acetic acid
with sulfuric acid, or a mixture of p-toluenesulfonic acid with acetic acid. There
is equally no particular limitation on the nature of the solvent, provided that it
has no adverse effect upon the reaction. Examples of preferred solvents include: alcohols,
such as methanol or ethanol; ethers, such as tetrahydrofuran or dioxane; and mixtures
of water with one or more of these organic solvents. The reaction will take place
over a wide range of temperatures, but we generally prefer to carry out the reaction
at a temperature within the range from 0 to 50°C. The time required for the reaction
will vary, depending upon many factors, notably the nature of the starting materials
and acids and the reaction temperature, but a period of from 10 minutes to 18 hours
will normally suffice.
[0058] Where an alkenyloxycarbonyl group is employed as the hydroxy-protecting group, it
may be removed by treating the compound with a base in the presence of an aqueous
solvent, under the same conditions as employed for removal of lower alkyl, aliphatic
acyl, aromatic acyl and alkoxycarbonyl groups. If the group is an allyloxycarbonyl
group, it can also simply be removed by using palladium and triphenylphosphine or
nickel tetracarbonyl, and this has the advantage that very few side reactions may
occur.
[0059] Depending upon the nature of the respective hydroxy-protecting groups and carboxy-protecting
groups, some of the reactions employed above may simultaneously remove carboxy-protecting
groups, as described below.
[0060] After completion of any of the above reactions, the desired compounds may be recovered
from the reaction mixture by conventional means, and, if required, may be further
purified by such conventional techniques as recrystallization or the various chromatography
techniques, particularly preparative thin layer chromatography or column chromatography.
[0061] Where a lower alkyl group is employed as a carboxy-protecting group, it may be removed
by treating the compound of formula (I) with a base in the presence of an aqueous
solvent. The reaction is carried out under the same conditions as are employed for
elimination of a hydroxy-protecting group, when that group is a lower alkyl, aliphatic
acyl, aromatic acyl or alkoxycarbonyl group; such groups may, therefore, be removed
simultaneously.
[0062] Where an aralkyl or lower haloalkyl group is employed as a carboxy-protecting group,
it may be removed by contacting the compound of formula - (I) with a reducing agent,
for example zinc in acetic acid. In the case of the aralkyl group, other reducing
agents include hydrogen in the presence of a catalyst and alkali metal sulfides (such
as potassium sulfide or sodium sulfide); such a reaction may be carried out under
the same conditions as are employed for removing a hydroxy-protecting group when that
group is an aralkyloxycarbonyl or aralkyl group; such a protecting group may, therefore,
be removed simultaneously.
[0063] Where an alkoxymethyl group is employed as the carboxy-protecting group, it may be
eliminated by treating the compound with an acid. The reaction is carried out under
the same conditions as are employed for eliminating a hydroxy-protecting group when
that group is an alkoxymethyl or substituted ethyl group; such a protecting group
may, therefore, be removed simultaneously.
[0064] After completion of these reactions, the desired compounds may be recovered from
the reaction mixture by conventional means. For example, one suitable recovery technique
comprises: filtering off insoluble materials, if any; washing the resulting organic
solvent solution containing the desired compound with water; drying the solution;
and then distilling off the solvent. The resulting residue may, if desired, be further
purified by various conventional techniques, such as recrystallization or the various
chromatography techniques, particularly preparative thin layer chromatography or column
chromatography.
[0065] The order in which the hydroxy-protecting groups and carboxy-protecting groups are
removed is not critical and these may be removed in any desired sequence or simultaneously.
[0066] Also, if desired, where the resulting product contains a free hydroxy and/or carboxy
group, one or more of these may be protected by a group which can easily be hydrolized
in vivo. This reaction may be carried out by conventional methods well-known to those
skilled in the art, the precise method chosen depending upon the nature of the protecting
group which is to be introduced.
[0067] For example, a carboxy group may be protected by conversion to the corresponding
ester capable of easy hydrolysis in vivo by reacting the acid with a halide corresponding
to the protecting group which it is desired to introduce, for example with: an aliphatic
acyloxymethyl halide, such as acetoxymethyl chloride, propionyloxymethyl bromide or
pivaloyloxymethyl chloride; a lower alkoxycarbonyloxyethyl halide, such as 1-methoxycarbonyloxyethyl
chloride or 1-ethoxycarbonyloxyethyl iodide; a phthalidyl halide, e.g. chloride, bromide
or iodide; or a (2-oxo-5-methyl-1,3-dioxolen-4-yl)-methyl halide, e.g. chloride, bromide
or iodide. The reaction is preferably effected in the presence of a solvent, the nature
of which is not crifical, provided that it has no adverse effect upon the reaction.
Preferred solvents are such polar solvents as dimethylformamide. The reaction will
take place over a wide range of temperatures, but we generally find it convenient
to carry out the reaction at a temperature in the range from 0 to 100°C. The time
required for the reaction may vary widely, depending upon many factors; however, under
the conditions suggested, a period of from 30 minutes to 10 hours will normally suffice.
[0068] The compounds of formula (11) used as starting materials in the process of the invention
may be prepared by a variety of reactions starting either from griseolic acid itself
[which has the formula (A) shown below] or dihydrodesoxygriseolic acid - [which has
the formula (B) shown below]:

[0069] As already described above, griseolic acid is a known compound disclosed, for example,
in European Patent Specification No. 29,329 or in US 'Patent Specification No. 4,460,765.
Dihydrodesoxygriseolic acid was disclosed in European Patent Publication No. 0162715,
published after the priority hereof. Both griseolic acid and dihydrodesoxygriseolic
acid may be produced by cultivating suitable microorganisms of the genus Streptomyces,
especially Stre
ptomvcesariseoaurantiacus SANK 63479 (deposited on 9th October 1979 at the Fermentation
Research Institue, Agency of Industrial 'Science and Technology, Japan, whence it
is available under the Accession No. FERM-P5223, and on 22nd October 1980 at the Agricultural
Research Service, Peoria, U.S.A., whence it is available under the Accession No. NRRL
12314). Full details of the characteristics of Streptomyces ariseoaurantiacus SANK
63479 are given in European Patent Publication No. 29,329A and in US Patent Specification
No. 4,460,765.
[0071] In the above formulae, R', R' and R
4 are as defined above. R
1a represents any of the groups defined above for R' and may be the same as or different
from R'. R
1b represents an acyl group, including any of the acyl groups hereinbefore defined in
relation to R'. R
8 represents an alkylsulfonyloxy group, a fluorinated lower alkylsulfonyloxy group
or an arylsulfonyloxy group, and examples of such groups are given previously in relation
to the same groups which may be represented by X. X
a represents a halogen atom, e.g. any one of those halogen atoms given previously in
relation to X.
[0072] The various reactions involved in this reaction - scheme may be carried out as described
below:
Protection of Carboxy Groups (Steps 1, 5 and 8)
[0073] In these reactions, the free carboxy groups of the griseolic acid of formula (A),
the dihydrodesoxygriseolic acid of formula (B) or the griseolic acid derivative of
formula (X) are, in Steps 1, 5 and 8, respectively, protected by protecting groups
R" and R
4, to give compounds of formulae (IV), (VIII) or - (XI) respectively. This reaction
may be effected by reacting the compond of formula (A), (B) or (X) with a diazo compound,
such as diazomethane or diphenyl diazomethane, or a p-tolyltriazene derivative, such
as N-methyl-p-tolyltriazene. The reaction is preferably effected in the presence of
a solvent, the nature of which is not critical, provided that it has no adverse effect
upon the reaction and that it is capable of dissolving, at least to some degree, the
starting materials employed in the reaction. Examples of suitable solvents include:
ketones, such as acetone; ethers, such as tetrahydrofuran; amides, such as dimethylformamide;
and mixtures of water with one or more of these organic solvents.
[0074] The reaction will take place over a wide range of temperatures, and there is no particular
limitation on the precise temperature chosen. We generally find it convenient to carry
out the reaction at a temperature in the range from -20°C to +50
°C. The time required for the reaction may vary widely, depending upon many factors,
notably the reaction temperature, but, for example, if the reaction is carried out,
as is preferred, at room temperature, a period of from 1 to 24 hours will normally
suffice.
[0075] If, instead of the free acids of formula (A), (B) or (X), the corresponding alkali
metal salts are employed, this protection step may be achieved by reacting the alkali
metal salt with a halide, for example methyl iodide, benzyl bromide, acetoxymethyl
bromide, 1-methoxycarbonyloxy iodide or (2-oxo-5-methyl-1,3-dioxolen-4-yl)methyl bromide,
by conventional means, to introduce the corresponding carboxy-protecting groups.
[0076] Protection of Hydroxy Groups (Steps 2 and 6)
[0077] In these reactions, the hydroxy groups of the compound of formula (IV) or the hydroxy
group of the compound of formula (VIII) may be protected by reacting the compound
with a corresponding acyl halide (such as acetyl chloride or benzoyl bromide) or a
corresponding acid anhydride (such as acetic anhydride) or with another halide corresponding
to the protecting group R' or R
1a (such as trimethylsilyl iodide) in the presence of a base to give the protected compound
of formula (V) or - (IX), respectively. The reaction is preferably effected in the
presence of a solvent, the nature of which is not critical, provided that it has no
adverse effect upon the reaction. In general, we prefer to use pyridine as the solvent,
as this has the advantage of also acting as the base.
[0078] The reaction will take place over a wide range of temperatures, although we generally
prefer to use a relatively low temperature, e.g. from -20°C to room temperature, in
order to control side reactions. The time required for the reaction will vary widely,
depending upon many factors, notably the reaction temperature; however, at temperatures
within the preferred range, a period of from 1 to 15 hours will normally suffice.
[0079] Alternatively, the compound of formula (IV) or - (VIII) can be reacted with an unsaturated
heterocyclic compound, such as dihydropyran, in the presence of an acid (e.g. hydrochloric
acid) to give a protected compound (V) or (IX) protected with a corresponding protecting
group.
Addition of a Hydrogen Halide Across the Double Bond (Step 3)
[0080] In this step, the compound of formula (V) is reacted with a hydrogen halide HX
a to add the hydrogen halide across the double bond and give the compound of formula
(VI). The reaction is preferably effected in the presence of a solvent, the nature
of which is not critical, provided that it has no adverse effect upon the reaction
and that it can dissolve the starting materials, at least to some degree. Preferred
solvents are organic acids, such as acetic acid. The hydrogen halide employed is preferably
hydrogen chloride, hydrogen bromide or hydrogen iodide. The reaction will take place
over a wide range of temperatures, e.g. from 0°C to 100°C, preferably either from
0°C to room temperature or from 80°C to 100°C. The time required for the reaction
will vary, depending upon many factors, notably the reaction temperature and the natures
of the solvents and reagents, but a period of from 1 to 72 hours will normally suffice.
[0081] If the hydroxy-protecting groups R' or R
1a are capable of removal under acidic conditions (e.g. the trimethylsilyl or tetrahydropyranyl
groups), they may be removed in the course of this step. In such a case, it may be
necessary or desirable to reinstate such protecting groups at the end of the step,
e.g. as described in relation to Steps 2 and 6.
Hydrodehalogenation (Step 4)
[0082] In this step, the halogen atom at the 4'-position of the compound of formula (VI)
is reduced to give the compound of formula (VII). This reaction may be carried out
either by using a tri-substituted tin hydride (such as tributyltin hydride) in an
aromatic hydrocarbon (such as benzene) or by using zinc powder in a lower aliphatic
carboxylic acid (such as acetic acid) or an alcohol (such as methanol or ethanol).
The reaction is preferably conducted either: using tributyltin hydride at the boiling
point of the solvent for a period of from 2 to 10 hours; or using zinc powder at a
temperature of from room temperature to 100°C for a period of from 2 to 20 hours
Selective Acylation of the 2'-Hydroxy Group (Step 7)
[0083] In this step, the hydroxy group at the 2'-position of griseolic acid (A) is selectively
acylated to give the compound of formula (X). This reaction may be effected either:
by adding an acylating agent to griseolic acid whilst the pH of the reaction solution
is maintained at a value of from 10 to 13 by means of a base, such as sodium hydroxide;
or by adding an acylating agent to a solution of griseolic acid in a buffer solution
of pH 10-13. The reaction is preferably effected in the presence of a solvent, the
nature of which is not critical, provided that it has no adverse effect upon the reaction;
water-immiscible solvents are preferred. The reaction will take place over a wide
range of temperatures, although we generally prefer to employ a temperature of from
-20°C to +
50
°C. The time required for the reaction may vary widely, depending upon many factors,
notably the reaction temperature and the natures of the solvents, bases and reagents,
but a period of from 1 to 10 hours will normally suffice.
Sulfonylation of the 7'-Hydroxy Group (Step 9)
[0084] In this step, the hydroxy group at the 7'-position of the compound of formula (XI)
is sulfonylated, to give the compound of formula (XII). The reaction is preferably
effected using a sulfonyl halide, such as methanesulfonyl chloride, p-toluenesulfonyl
chloride or trifluoromethanesulfonyl chloride, in the presence of an acid-binding
agent, such as pyridine or dimethylaminopyridine. The reaction is preferably effected
in the presence of a solvent, the nature of which is not critical, provided that it
has no adverse effect upon the reaction. Preferred solvents are halogenated hydrocarbons,
especially halogenated aliphatic hydrocarbons, such as methylene chloride or chloroform.
The reaction will take place over a wide range of temperatures, although we generally
prefer to employ a relatively low temperature, e.g. from -10°C to room temperature.
in order to control side reactions. The time required for the reaction may vary widely,
depending upon many factors, notably the reaction temperature and the natures of the
reagents, but a period of from 1 to 20 hours will normally suffice.
Reduction of 7'-Sulfonyloxy Group (Step 10)
[0085] In this reaction, the compound of formula (XIII) is prepared by replacing the sulfonyloxy
group at the 7'-position of the compound of formula (XII) by a halogen atom and then
replacing this halogen atom by a hydrogen atom.
[0086] Replacement of the sulfonyloxy group by a halogen atom is effected by reacting the
compound of formula (XII) with an anhydrous lithium halide. The reaction is preferably
effected in the presence of a solvent, the nature of which is not critical, provided
that it has no adverse effect upon the reaction. Examples of preferred solvents are
such polar solvents as: acid amides, such as dimethylformamide or hexamethylphosphoric
triamide; sulfoxides, such as dimethyl sulfoxide; and alkyl phosphates, such as triethyl
phosphate. The reaction will take place over a wide range of temperatures and the
precise temperature chosen is not critical. We generally find it convenient to employ
a temperature in the range from 0 to 150°C. The time required for the reaction may
vary, depending on many factors, notably the temperature and nature of the reagents;
however, under the conditions suggested above, a period of from 1 to 10 hours will
normally suffice.
[0087] In a next stage, the halogen atom is replaced by a hydrogen atom. This may be effected
by means of any conventional reducing - (hydrogenating) agent capably of replacing
a halogen atom by a hydrogen atom. It is preferably effected using zinc/acetic acid
and may be carried out by adding zinc powder to a solution of the halogenated compound
of formula (XII) in aqueous acetic acid. The reaction will take place over a wide
range of temperatures, although a temperature from 0°C to 150°C is preferred. The
time required for the reaction will vary, depending upon many factors, notably the
reaction temperature and the nature of the reagents, but a period of from 1 to 10
hours will normally suffice.
[0088] In addition, if desired, the group R
lb may be removed by conventional alkaline hydrolysis (for example with sodium hydroxide
in aqueous methanol) and then the resulting hydroxy group may, if desired, be re-protected,
as described in Step 2.
[0089] The process of the present invention is further illustrated by the following Examples.
Of the starting materials, griseolic acid may be prepared as described in aforementioned
US Patent Specification No. 4,460,765, dimethyl griseolate may be prepared by simple
esterification of griseolic acid - (e.g. as described in Steps 1, 5 and 8 above) and
dihydrodesoxygriseolic acid may be prepared as described in the subsequent Preparation.
EXAMPLE 1
[0090] N
6 -Methylgriseolic acid
(a) 2 ml of methyl iodide were added to a solution of 1.63 g of dimethyl griseolate
in 20 ml of dimethylformamide, and the mixture was stirred at room temperature for
48 hours in a sealed vessel. The solvent was then evaporated off under reduced pressure,
and the residue was mixed with 10 ml each of acetone and toluene, and then the solution
was concentrated by evaporation under reduced pressure. This operation was repeated
a total of three times to give a residue, which was dissolved in 30 ml of water and
adjusted to a pH value of 5.7 with a 0.1 N aqueous solution of sodium hydroxide. The
mixture was heated at 100°C for 2.5 hours, while at 30 minutes intervals the pH was
re-adjusted to 5.7. The reaction mixture was then evaporated under reduced pressure
to reduce the volume to 10 ml. 10 ml of a 2N aqueous solution of sodium hydroxide
were added to the concentrate and the mixture was allowed to stand for 2 hours. The
mixture was then adjusted to a pH value of 2.3 and purified by chromatography using
an RP-8 prepacked column (Merck). The main fractions were collected, lyophilized and
recrystallized from water, to give 690 mg of the title compound.
(b) 25 ml of methyl iodide were added to a solution of 18.95 g of griseolic acid in
230 ml of dimethylformamide, and the mixture was stirred at room temperature for 42
hours in a sealed vessel. At the end of this time, the solution was concentrated by
evaporation under reduced pressure. The residue was then mixed with 70 ml each of
ethanol and toluene and again concentrated by evaporation under reduced pressure.
This operation was repeated a total of four times to give a residue, to which 300
ml of ethanol were added. The mixture was again cancentrated by evaporation under
reduced pressure to give a powdery residue. A suspension of the residue in 800 ml
of ethyl acetate was ultrasonically treated to give a powder, and was then allowed
to stand overnight in a refrigerator. Filtration of the suspension yielded 272 g of
a pale yellow powder. The powder was dissolved in a 1 N aqueous solution of sodium
hydroxide, and adjusted to a pH value of 7.0. Water was added to the solution to give
a total volume of 200 ml. A mixture of the solution with 50 ml of a 0.5M phosphate
buffer (pH 7.0) was heated for 3 hours under reflux, and then the pH of the mixture
was adjusted with a 1N aqueous solution of sodium hydroxide to a value of 7.0. The
mixture was heated under reflux for 2 hours, and then its pH was adjusted with concentrated
hydrochloric acid to a value of 2.3. It was then treated with activated carbon, concentrated
by evaporation under reduced pressure to an amount of about 130 ml, and then subjected
to column chromatography using an RP-8 prepacked column (Merck), eluted with water
containing 3% v/v acetonitrile. The main fractions were collected, concentrated and
recrystallized from water to give 12.73 g of the title compound. Concentration and
cooling of the mother liquor yielded an additional 2.01 g of the title compound.
[0091] Ultraviolet Absorption Spectrum (H
20) X
maxnm - (E):
265 (17200).
[0092] Nuclear Magnetic Resonance Spectrum [(CD3)-2S0] 205 ppm:

N
6 -Methyl-7' -desoxy-4'a,5'-dihydrogriseolic acid
[0093] 1 ml of methyl iodide was added to a solution of 100 mg of 7'-desoxy-4'a,5'-dihydrogriseolic
acid in 20 ml of dimethylformamide, and the mixture was allowed to stand at room temperature
for 24 hours in a sealed vessel. The solvent was distilled off under reduced pressure
to give a residue. 10 ml each of acetone and toluene were added to the residue and
the mixture was concentrated by evaporation under reduced pressure. This operation
was repeated twice. A solution of the resulting residue in 20 ml of a 0.5M phosphate
buffer of pH 7.0 was stirred for 3 hours under reflux, to give a reaction mixture
which was purified by column chromatography using an RP-8 prepacked column (Merck),
eluted with water containing 3% v/v acetonitrile, followed by lyophilizing the main
fractions to give 67 mg of the title compound as a white powder.
[0094] Ultraviolet Absorption Spectrum (∈) λ
max:

[0095] Nuclear Magnetic Resonance Spectrum [(CD
3)-
2SO+D
2O] 8 ppm:

EXAMPLE 3
[0096] N
6 -Methylgriseolic acid 6 ml of methyl iodide and 250 mg of sodium sulfite were added
to a solution of 3.8 g of griseolic acid in 50 ml of dimethylformamide, and the mixture
was heated at 40°C for 17 hours, whilst stirring. The solvent was then distilled off
under reduced pressure to give a residue. 20 ml each of ethanol and toluene were added
to the residue, and the mixed solvent was distilled off under reduced pressure. This
operation was repeatd three times. 50 ml of a 1N aqueous solution of sodium hydroxide
were added to the residue and the mixture was allowed to stand at room temperature
for 4 days. The mixture was then adjusted to a pH value of 2.3 with concentrated hydrochloric
acid and allowed to stand for 1.5 hours whilst ice-cooling. The crystals which precipitated
were collected by filtration, to yield 3.0 g of the title compound as crude crystals.
The filtrate was then subjected to column chromatography using 200 ml of a CHP-20
resin. After washing with 1.5 litre of water, the main fractions were eluted with
water containing 3% v/v acetonitrile, followed by lyophilizing to give 0.9 g of the
title compound. The above crude crystals and the substance lyophilized were together
dissolved in 20 ml of a 1N aqueous solution of sodium hydroxide and the mixture was
weakly acidified with 5 ml of a 1 N aqueous solution of hydrochloric acid, treated
with active carbon, and then further acidified with 15 ml of a 1 N aqueous solution
of hydrochloric acid. It was then allowed to stand at 5°C overnight. The crystals
which precipitated were collected and dried over phosphorus pentoxide to give 2.90
g of the title compound, whose priorities were the same as those of the product of
Example 1.
EXAMPLE 4
[0097] N6 -Methyt-7'-desoxy-4'α,5'-dihydrogriseolic acid
[0098] 1 ml of methyl iodide and 7 mg of sodium sulfite were added to a solution of 100
mg of 7'- desoxy-4'a,5'-dihydrogriseolic acid in 20 ml of dimethylformamide, and the
mixture was heated at 40°C for 17 hours, whilst stirring. The solvent was then distilled
off under reduced pressure to give a residue. 10 ml each of acetone and toluene were
added to the residue and the mixed solvent was distilled off under reduced pressure.
This operation was repeated three times. 1.5 ml of a 1 N aqueous solution of sodium
hydroxide were added to the residue and the mixture was allowed to stand at room temperature
for 4 days. The mixture was then adjusted to a pH value of 2.3 with concentrated hydrochloric
acid and purified by chromatography using an RP-8 prepacked column (Merck) (eluent
: water containing 3% v/v acetonitrile). The main fractions were collected and lyophilized
to give 80 mg of the title compound as a white powder, whose properties were the same
as those of the product of Example 2.
PREPARATION
[0099] Dihydrodesoxygriseolic acid
[0100] 30 litres of a medium having a pH of 7.0 before sterilization and the following composition
- (percentages are w/v) were prepared:

Water to 100%
[0101] 15 litres of this medium were charged into each of two 30 litre jar fermenters, which
were then sterilized under pressure at 120°C for 30 minutes. The culture medium was
cooled, and then 150 ml - (1 % by volume) of a culture broth of Stre
ptomvces ariseoaurantiacus SANK 63479 (which had previously been incubated in the medium
described above by means of a rotatory shaking cultivator at 28°C for 72 hours) were
inoculated into each fermenter. Cultivation was then carried out at 28°C for 96 hours
under aeration at the rate of 15 litres per minute and with agitation at the rate
of 200 rpm.
[0102] The two culture broths were then filtered to remove the mycelial cake and the combined
filtrates (pH 7.0), in a total volume of 28 litres, were passed through a column of
Diaion HP 20 ( a trademark for an ion-exchange resin produced by Mitsubishi Chemical
Industries Ltd.) and then adsorbed on a column of activated charcoal. This column
was washed with water and then the adsorbed material was eluted with a 60:40 by volume
mixture of acetone and water. The acetone was evaporated from the resulting solution
under reduced pressure and the remaining aqueous solution was concentrated by evaporation
under reduced pressure and then lyophilized, to give 150 mg of a crude powder.
[0103] This crude powder was dissolved in a small amount of distilled water and then adsorbed
on Dowex 1 x 4 (CI - form, a trademark for an ion-exchange resin produced by the Dow
Chemical Company). At this stage, the product was a mixture of griseolic acid and
dihydroesoxygriseolic acid. This mixture was subjected to gradient elution with a
sodium chloride gradient to separate the two components and then the eluate was subjected
to column chromatography through Sephadex LH-20 - (a trademark for a product of Pharmacia
Co) and the dihydrodesoxygriseolic acid was eluted with water. The fractions containing
this substance were combined and their pH was adjusted to a value of 2.5 by the addition
of 1N aqueous hydrochloric acid. The product was then adsorbed on a column of Diaion
HP 20, washed with water and then eluted with a 60:40 by volume mixture of acetone
and water. The eluate was left to stand overnight at 4°C, whereupon the dihydrodesoxygriseolic
acid separated out as plates. These were separated from the liquor, giving a total
of 1.87 mg of dihydrodesoxy griseolic acid, as white plates melting at 160°C (with
decomposition, accompanied by a brown discoloration). This compound gave a single
spot on silica gel thin layer chromatography - (silica gel Art. 5715, a product of
Merck & Co. Inc.).